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Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

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Growth kinetics of S. cerevisiae ISC mutants in the presence of ROS generators.A–E) Yeast cultures were grown on YPD liquid medium as follows: A) no addition, B) H2O2 6.25 mM, C) H2O2 12.5 mM, D) menadione 80 µM, E) menadione 150 µM, and F) ethanol 8% (v/v). Cultures were incubated at 30°C with light shaking and growth (biomass) was determined by measuring O.D. at 600 nm. Values representing the mean and standard errors of the mean (SEM) are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants versus controls. Significant differences (p<0.05) are indicated with (*).
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pone-0111585-g001: Growth kinetics of S. cerevisiae ISC mutants in the presence of ROS generators.A–E) Yeast cultures were grown on YPD liquid medium as follows: A) no addition, B) H2O2 6.25 mM, C) H2O2 12.5 mM, D) menadione 80 µM, E) menadione 150 µM, and F) ethanol 8% (v/v). Cultures were incubated at 30°C with light shaking and growth (biomass) was determined by measuring O.D. at 600 nm. Values representing the mean and standard errors of the mean (SEM) are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants versus controls. Significant differences (p<0.05) are indicated with (*).

Mentions: In order to verify whether the dysfunction in the ISC system is related to a parallel increase in the sensitivity to oxidative damage, the susceptibility to several ROS-generating compounds was tested using three ISC mutants, whose disrupted genes encode proteins that play important roles in assembly of Fe–S centers. In addition, control S. cerevisiae strains that display a severe imbalance in iron homeostasis were used; these included atx1Δ mutants, which are impaired in high affinity iron-depleted medium, as this gene is involved in copper trafficking and delivery to Fet3p, which oxidizes Fe2+ to Fe3+ for uptake by Ftr1p [31] and mrs4Δ mutants, which show cellular iron accumulation and sensitivity to H2O2 and menadione, as this gene is co-regulated with the iron regulon, and encodes the Mrs4 Fe2+ iron transporter at the inner mitochondrial membrane under conditions of iron deprivation [15]–[17]. aft1Δ mutant shows increased ROS sensitivity and iron accumulation by inducing iron-sensing genes under iron depletion conditions [32]. All these yeast strains show iron-dependence when grown in the presence of phenanthroline, which induces iron-depletion, and which was improved with iron addition in the culture media (Figure S1). aft1Δ mutant is more sensitive to iron depletion than mrs4Δ mutant, which in turn correlates with the exacerbated sensitivity of the aft1Δ mutant to ROS inducers (Fig. 1). Of the ISC mutants, ssq1Δ, grx5Δ, and isa1Δ mutants showed a significantly impaired growth rate compared to WT, displaying severely compromised growth at concentrations in the range of 6.25–12.5 mM H2O2. grx5Δ mutants were the least sensitive at all concentration of H2O2 (Fig. 1b–c). The susceptibility to menadione (a superoxide generator), followed a similar pattern to that observed with H2O2 treatments: at 80 µM menadione, ISC mutants showed a moderate but significant inhibition in their growth kinetics, with respect to the WT strain (Fig. 1d), while at 150 µM menadione, also with ethanol (8%), the growth of all ISC mutants was drastically affected (Fig. 1e–f); again, grx5Δ was the least sensitive ISC mutant to the stressor. As expected, atx1Δ and mrs4Δ mutants (which show imbalanced iron homeostasis), showed similar behavior to the WT under iron sufficiency, except in YPD supplemented with 12.5 mM H2O2 (Fig. 1c), in which delayed growth was observed. In contrast, the hypersensitive aft1Δ mutant exhibited a marked sensitivity to H2O2, menadione, and ethanol treatments (Fig. 1b–f).


Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Growth kinetics of S. cerevisiae ISC mutants in the presence of ROS generators.A–E) Yeast cultures were grown on YPD liquid medium as follows: A) no addition, B) H2O2 6.25 mM, C) H2O2 12.5 mM, D) menadione 80 µM, E) menadione 150 µM, and F) ethanol 8% (v/v). Cultures were incubated at 30°C with light shaking and growth (biomass) was determined by measuring O.D. at 600 nm. Values representing the mean and standard errors of the mean (SEM) are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants versus controls. Significant differences (p<0.05) are indicated with (*).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214746&req=5

pone-0111585-g001: Growth kinetics of S. cerevisiae ISC mutants in the presence of ROS generators.A–E) Yeast cultures were grown on YPD liquid medium as follows: A) no addition, B) H2O2 6.25 mM, C) H2O2 12.5 mM, D) menadione 80 µM, E) menadione 150 µM, and F) ethanol 8% (v/v). Cultures were incubated at 30°C with light shaking and growth (biomass) was determined by measuring O.D. at 600 nm. Values representing the mean and standard errors of the mean (SEM) are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants versus controls. Significant differences (p<0.05) are indicated with (*).
Mentions: In order to verify whether the dysfunction in the ISC system is related to a parallel increase in the sensitivity to oxidative damage, the susceptibility to several ROS-generating compounds was tested using three ISC mutants, whose disrupted genes encode proteins that play important roles in assembly of Fe–S centers. In addition, control S. cerevisiae strains that display a severe imbalance in iron homeostasis were used; these included atx1Δ mutants, which are impaired in high affinity iron-depleted medium, as this gene is involved in copper trafficking and delivery to Fet3p, which oxidizes Fe2+ to Fe3+ for uptake by Ftr1p [31] and mrs4Δ mutants, which show cellular iron accumulation and sensitivity to H2O2 and menadione, as this gene is co-regulated with the iron regulon, and encodes the Mrs4 Fe2+ iron transporter at the inner mitochondrial membrane under conditions of iron deprivation [15]–[17]. aft1Δ mutant shows increased ROS sensitivity and iron accumulation by inducing iron-sensing genes under iron depletion conditions [32]. All these yeast strains show iron-dependence when grown in the presence of phenanthroline, which induces iron-depletion, and which was improved with iron addition in the culture media (Figure S1). aft1Δ mutant is more sensitive to iron depletion than mrs4Δ mutant, which in turn correlates with the exacerbated sensitivity of the aft1Δ mutant to ROS inducers (Fig. 1). Of the ISC mutants, ssq1Δ, grx5Δ, and isa1Δ mutants showed a significantly impaired growth rate compared to WT, displaying severely compromised growth at concentrations in the range of 6.25–12.5 mM H2O2. grx5Δ mutants were the least sensitive at all concentration of H2O2 (Fig. 1b–c). The susceptibility to menadione (a superoxide generator), followed a similar pattern to that observed with H2O2 treatments: at 80 µM menadione, ISC mutants showed a moderate but significant inhibition in their growth kinetics, with respect to the WT strain (Fig. 1d), while at 150 µM menadione, also with ethanol (8%), the growth of all ISC mutants was drastically affected (Fig. 1e–f); again, grx5Δ was the least sensitive ISC mutant to the stressor. As expected, atx1Δ and mrs4Δ mutants (which show imbalanced iron homeostasis), showed similar behavior to the WT under iron sufficiency, except in YPD supplemented with 12.5 mM H2O2 (Fig. 1c), in which delayed growth was observed. In contrast, the hypersensitive aft1Δ mutant exhibited a marked sensitivity to H2O2, menadione, and ethanol treatments (Fig. 1b–f).

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Show MeSH